Abstract/Project Summary Recent human genetic studies have shown that loss or gain of function variants of the voltage-gated potassium channel KCNQ2 causes neonatal epileptic encephalopathy. It is currently assumed that KCNQ2 channels primarily associate with KCNQ3 channels in the brain; however, several studies have suggested that KCNQ2 channels may also interact with additional transmembrane proteins. Identifying the KCNQ2 membrane complex in the brain is necessary in order to understand how KCNQ2 channels dysfunction could lead to epilepsy and to also design better therapeutics. To address this question, we have developed a new epitope tagged mouse line allowing us to analyze KCNQ2 affinity purified complexes from the hippocampus and neocortex using mass spectrometry. Indeed, our preliminary data show that KCNQ2 channels could associate with KCNQ3 as well as KCNQ5 channels. This finding goes against the current dogma that KCNQ2 only associates with KCNQ3 channels in the nervous system. Therefore, in this proposal we plan to examine whether native KCNQ2 complex channels in the brain are comprised of KCNQ3 and KCNQ5 channels. To this end, we will: (i) establish the presence of KCNQ2/5 or KCNQ2/3/5 complexes in the brain and (ii) probe the biophysical properties of KCNQ2/5-containing KCNQ channels. The proposed research will make a significant contribution to our broader understanding of how KCNQ2, KCNQ3, and KCNQ5 channels control neuronal excitability, and build a foundation for preventing and treating disorders associated with neuronal KCNQ dysfunction.